Kv1 channels are fundamental components of neuronal signaling through effects on neuronal excitability and synaptic transmission. This proposal is aimed at determining the fundamental mechanisms that govern expression and localization of Kv1 channels in mammalian hippocampus, specifically in axons and nerve terminals of the perforant path. Using novel, state-of-the-art mass spectrometric approaches we have made great inroads in defining in vivo phosphosites on brain Kv1.2 and Kv22 subunits, and find that many are Pro- associated pSer and conform to consensus binding sites for proteins containing pSer binding modules. Phosphorylation at some of these sites is specific to channels in axons and nerve terminals, and phosphorylation at these sites changes in response to epileptic seizures. Moreover, these sites are likely phosphorylated by proline-directed kinases (ProDKs), whose importance in neuronal function and as targets for new therapeutics is just now being appreciated. These data provide the first opportunity to investigate the role of bona fide and unambiguously identified in vivo brain phosphosites on Kv1 channel subunits in governing their expression levels and subcellular localization in hippocampus. We will test the overall hypothesis of this proposal that these sites are crucial to neuronal function and plasticity as mediated by ProDKs acting on native Kv1 channels. In aims 1-2 we will accomplish this by examining the effects of interventions that alter the phospho-state of Kv1.2 and Kv22 subunits. We will mutate identified in vivo ProDK phosphorylation sites, and candidate upstream ProDK priming sites, and intervene in ProDK expression levels in heterologous cells and hippocampal neurons and determine effects on Kv1 channel expression and localization. These experiments will provide insights into the specific role of these in vivo sites in regulating Kv1 channel biology. In aim 3 we will define the effects of epileptic seizures on Kv1.2 and Kv22 phosphorylation, relative to changes in perforant path function in response to acute seizures and during the acquisition of spontaneous recurrent seizures. We will also define the precise cellular and subcellular locations of ProDK-phosphorylated Kv1.2 and Kv22 in normal and epileptic hippocampus. In aim 4 we will identify cellular proteins exhibiting phospho-dependent interaction with Kv1.2 and Kv22 and determine their role in expression and localization. These studies will yield important insights into the physiological and pathological regulation of Kv1 channels, which are key regulators of neuronal excitability and synaptic transmission in mammalian hippocampus. PUBLIC HEALTH RELEVANCE: This study aims to better understand basic mechanisms controlling brain function. It focuses on neuronal ion channels and their regulatory enzymes that are important targets for developing new therapeutics for epilepsy.